Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.


Introduction
Intracellular nucleotide-binding and leucine-rich repeat (NLR) domain-containing immune receptors are essential components of the plant innate immune system (Jones and Dangl, 2006;Jones et al., 2016). These receptors detect effector proteins which are delivered into host cells by invading pathogens and pests to promote virulence. NLR receptors have a modular domain architecture typically consisting of an N-terminal coiled-coil (CC or CC R ) or Toll/Interleukin-1 receptor (TIR) domain, a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR) domain. In addition, some NLRs contain non-canonical domains which are integrated into the protein architecture either at the N-or C-termini or between canonical domains (Sarris et al., 2016;Cesari et al., 2014;Kroj et al., 2016;Bailey et al., 2018). These integrated domains (IDs) resemble effector virulence targets and either directly bind or are modified by effector proteins to activate NLR-mediated immune signaling (Cesari et al., 2014;Oikawa et al., 2020;Maidment et al., 2021;Maqbool et al., 2015;Sarris et al., 2015;Mukhi et al., 2021).
Many of the characterized resistance (R) genes used to confer disease resistance in crop breeding programs encode NLR proteins (Kourelis and van der Hoorn, 2018). However, NLR-mediated resistance can be overcome through silencing or deletion of effectors in pathogen genomes, the gain of new effectors or effector functions, or mutation to evade NLR activation (Raffaele et al., 2010;Yoshida et al., 2016). Engineering NLRs to detect currently unrecognized effector proteins would provide new opportunities to control plant pathogens. Early attempts to engineer NLRs focused on random mutagenesis followed by gain-of-function screening, with some success in both expanding recognition profiles to new effector variants and increasing the sensitivity of the receptor (Giannakopoulou et al., 2015;Segretin et al., 2014;Farnham and Baulcombe, 2006;Harris et al., 2013). More recently, modification of the effector target PBS1, which is guarded by the NLR protein RPS5 led to successful engineering of novel recognition by this system (Kim et al., 2016;Helm et al., 2019). RPS5 is activated by cleavage of the Arabidopsis thaliana protein kinase PBS1 by the Pseudomonas syringae effector AvrPphB. By varying the PBS1 cleavage site, the RPS5/PBS1 system has been engineered to recognize proteases from different pathogens Helm et al., 2019). Using a different strategy, a protein domain targeted for degradation by the phytoplasma effector SAP05 was fused to the C-terminus of the TIR-NLR RRS1-R. RRS1-R represses the immune cell death-triggering activity of a second TIR-NLR, RPS4. While transient co-expression of the engineered RRS1-R, RPS4, and the phytoplasma effector SAP05 led to cell death in N. tabacum, transgenic A. thaliana plants were not resistant to phytoplasma carrying the SAP05 effector .
Integrated domains can facilitate the recognition of structure-and sequence-diverse effectors which target similar host proteins. This is exemplified by the TIR-NLR pair RRS1 and RPS4, which mediate recognition of the structurally distinct effectors PopP2 from Ralstonia solanacearum and AvrRps4 from Pseudomonas syringae pv. pisi Mukhi et al., 2021;Deslandes et al., 2003;Saucet et al., 2015;Huh et al., 2017;Le Roux et al., 2015). Furthermore, the potential to replace naturally occurring integrated domains with nanobodies of defined specificity to confer disease resistance has recently been demonstrated (Kourelis et al., 2023). Modification of existing integrated domains, or the incorporation of entirely new protein domains into an NLR structure could deliver new recognition specificities and extend the toolbox of resistance genes available to combat crop pathogens and pests.
The paired rice CC-NLR proteins Pik-1 and Pik-2 cooperatively activate plant defense in response to the blast pathogen effector AVR-Pik (Ashikawa et al., 2008;Kanzaki et al., 2012;Zdrzałek et al., 2020). The sensor NLR Pik-1 contains an integrated HMA domain between the CC and NB-ARC domains (Maqbool et al., 2015, Figure 1-figure supplement 1a). Direct binding of AVR-Pik to the HMA domain is required to activate Pik-mediated immunity (Maqbool et al., 2015;De la Concepcion et al., 2018;De la Concepcion et al., 2021). Multiple Pik alleles have been described in different rice cultivars, with most amino acid polymorphisms located within the integrated HMA domain of Pik-1. Five Pik alleles (Pikp, Pikm, Pikh, Piks, and Pik*) have been functionally characterized for their response to blast isolates carrying different AVR-Pik variants (Maqbool et al., 2015;Kanzaki et al., 2012;De la Concepcion et al., 2018;De la Concepcion et al., 2021, Figure 1-figure supplement 1c). To date, six AVR-Pik variants (A-F) have been described, which differ in five amino acid positions at the HMAbinding interface (Kanzaki et al., 2012;Yoshida et al., 2009;Longya et al., 2019, Figure 1-figure OsHIPP19 share 51% amino acid identity and are structurally similar; the RMSD (as calculated in Coot using secondary structure matching) between Pikp-HMA (PDB 6G10) and OsHIPP19-HMA (PDB 7B1I) is 0.97 Å across 71 amino acids. We hypothesized that exchanging the HMA domain of Pikp-1 for the HMA domain of OsHIPP19 would result in an NLR capable of binding and responding to AVR-PikC and AVR-PikF.
For this exchange, amino acids 188-263 (inclusive) of Pikp-1 were replaced with amino acids 2-77 of OsHIPP19 to give the chimeric NLR protein Pikp-1 OsHIPP19 (Figure 1-figure supplement 2a). To test whether Pikp-1 OsHIPP19 could associate with AVR-Pik effector variants in planta, we performed co-immunoprecipitation experiments in N. benthamiana. Each of the myc-AVR-Pik variants (an N-terminal myc tag was used for effectors in all experiments in N. benthamiana) was transiently co-expressed with Pikp-1 OsHIPP19 -HF (C-terminal 6xHis/3xFLAG tag, used for all  by agroinfiltration. Pikp-2 was not included to prevent the onset of cell death, which reduces protein levels in the plant cell extract, and previous work has shown that AVR-Pik can associate with Pik-1 in the absence of Pik-2 (De la Concepcion et al., 2018). Following immunoprecipitation with anti-FLAG beads to enrich for Pikp-1 OsHIPP19 , western blot analysis showed that all AVR-Pik variants, except AVR-PikF, co-precipitated with Pikp-1 OsHIPP19 (Figure 1-figure supplement 3). As a control, and consistent with previous studies, AVR-PikD associated with Pikp-1, while AVR-PikC did not.

Structure-guided mutagenesis of Pikp-1 extends response to previously unrecognized AVR-Pik variants
Alongside the HMA-domain exchange strategy, we also used a structure-guided approach to target point mutations in Pikp-HMA that could extend the effector recognition profile of Pikp without triggering autoimmunity.

Figure 1 continued on next page
Other leaf positions represent relevant negative controls. (B) Pikp-mediated response scoring represented as dot plots to summarize 30 repeats of the experiment shown in (A) across three independent experiments (Materials and methods, Figure 1-figure supplement 4a). Fluorescence intensity is scored as previously described (Maqbool et al., 2015;De la Concepcion et al., 2018). (C) The Pikp-1 OsHIPP19-mbl7 chimera does not display autoactive cell death in N. benthamiana (bottom right), as seen for Pikp-1 OsHIPP19 (middle left), but retains response to AVR-PikD and expands Pikp-mediated response to AVR-PikC (middle right). (D) Pikp-mediated response scoring represented as dot plots to summarize 60 repeats of the experiment shown in (C) across three independent experiments (Materials and methods, Figure 1-figure supplement 4b). (E) As for (C), but showing the expanded Pikpmediated response to AVR-PikF. (F) As for (D) but for 60 repeats of the experiment in (E) across three independent experiments (Materials and methods, Figure 1-figure supplement 4c). (G) Western blots following co-immunoprecipitation reveal that the Pikp-1 OsHIPP19-mbl7 chimera associates with all AVR-Pik effector variants in N. benthamiana. Plant cell lysates were probed for the expression of Pikp-1/Pikp-1 OsHIPP19-mbl7 and AVR-Pik effector variants using anti-FLAG and anti-Myc antiserum, respectively. Total protein extracts were visualized by Ponceau Staining.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Unedited and uncropped blot for panel G, IP Pik-1 α-FLAG, Pik-1 α-FLAG, with relevant bands labeled.
Source data 9. Unedited and uncropped blot for panel G, Ponceau stain, with relevant bands labeled.
Source data 10. Unedited and uncropped blot for panel G, Ponceau stain.
Source data 11. Cell death scores used for dot plots in panel B.
Source data 12. Cell death scores used for dot plots in panel D.
Source data 13. Cell death scores used for dot plots in panel F.                       De la Concepcion et al., 2019). These interfaces are also observed in the structure of the HMA domain of OsHIPP19 in complex with AVR-PikF (PDB accession code 7B1I Maidment et al., 2021). The Asp67 and Lys78 polymorphisms that distinguish AVR-PikC and AVR-PikF from AVR-PikE and AVR-PikA, respectively, are located at interface 2. In the crystal structure of Pikh-HMA/AVR-PikC, the side chain of AVR-PikC Asp67 extends towards a loop in the HMA domain containing Pikh-HMA Asp224 . This loop is shifted away from the effector, likely due to steric clash and/or repulsion by the two Asp sidechains, and intermolecular hydrogen bonds between Pikh-HMA Asp224 and AVR-PikC Arg64 are disrupted. We hypothesized that compensatory mutations at interface 2 could mitigate against the disruption caused by AVR-PikC Asp67 . Therefore, we introduced Asp224Ala and Asp224Lys mutations in the Pikp-1 NK-KE background (De la Concepcion et al., 2019) and tested these constructs in cell death assays in N. benthamiana. Neither mutation extended the Pikp-1 NK-KE -mediated cell death response to AVR-PikC, and both mutations reduced the extent of the response to AVR-PikD (                                 Pikh-1 and Pikp-1 NK-KE differ from Pikp-1 by one and two amino acids, respectively, at interface 3. These amino acid differences are sufficient to extend binding and cell death response to AVR-PikE and AVR-PikA, even though the residues that distinguish these variants from AVR-PikD are located at interface 2. We, therefore, predicted that we could engineer a modified Pik-1 that interacts with AVR-PikC/AVR-PikF by mutating other interfaces in the HMA domain to compensate for disruption at the site of the polymorphic residue. The crystal structure of the OsHIPP19-HMA/AVR-PikF complex revealed additional hydrogen bond interactions at interface 3 relative to integrated HMAs in the complex with AVR-Pik variants . The side chain of OsHIPP19 Glu72 was particularly striking. The corresponding residue in all described Pik-1 HMA domains is serine, and while the hydroxyl group of the serine side chain only forms an intramolecular hydrogen bond within the HMA domain, the bulkier OsHIPP19 Glu72 side chain extends across the interface and forms a direct hydrogen bond with the effector (Figure 2a). We, therefore, introduced a Ser258Glu mutation in the Pikp-1 NK-KE background to give the triple mutant Pikp-1 SNK-EKE and tested the ability of this protein to respond to AVR-Pik variants in N. benthamiana cell death assays. Firstly, we confirmed that Pikp-1 SNK-EKE was not autoactive, evidenced by a lack of cell death following co-expression with Pikp-2 only (in the absence of effectors). Next, we established that Pikp-1 SNK-EKE remained functional and caused cell death on co-expression with Pikp-2 and AVR-PikD. Crucially, transient expression of Pikp-1 SNK-EKE , but not Pikp-1 NK-KE , with Pikp-2 and either AVR-PikC or AVR-PikF, triggered cell death suggestive of new effector response specificities ( We tested whether the Ser258Glu mutation alone was sufficient to extend the cell death response to AVR-PikC or AVR-PikF using the cell death assay. When Pikp-1 S258E was co-infiltrated with Pikp-2 and either AVR-PikC or AVR-PikF no cell death was observed (Figure 2-figure supplement 8, Figure 2figure supplement 9), demonstrating that the triple mutation is necessary for response to these effectors.

The Ser258Glu mutation extends the binding of Pikp-HMA NK-KE to AVR-PikC and AVR-PikF in vitro
The extent of the Pik/AVR-Pik-dependent cell death response in N. benthamiana largely correlates with binding affinity in vitro and in planta (Maqbool et al., 2015;De la Concepcion et al., 2018;De la Concepcion et al., 2021;De la Concepcion et al., 2019). To test whether the Pikp-1 SNK-EKE response to AVR-PikC or AVR-PikF in N. benthamiana correlates with increased binding to the modified HMA domain, we first used surface plasmon resonance (SPR) with purified proteins. Pik-HMA domains and AVR-Pik effectors were purified from E. coli cultures using established protocols for the production of these proteins Maqbool et al., 2015;De la Concepcion et al., 2018;De la Concepcion et al., 2021;De la Concepcion et al., 2019). For SPR, AVR-Pik effector variants were immobilized on a Ni 2+ -NTA sensor chip via a C-terminal 6xHis tag. Pikp-HMA, Pikp NK-KE -HMA or Pikp SNK-EKE -HMA has flowed over the surface of the chip at three different concentrations (4 nM, 40 nM, and 100 nM). The binding (R obs , measured in response units, RU) was recorded and expressed as a percentage of the maximum theoretical responses (%Rmax), assuming a 2:1 HMA:effector interaction model (Maqbool et al., 2015). Consistent with previous studies, Pikp-HMA did not bind AVR-PikC (nor AVR-PikF), and weak binding was observed for Pikp NK-KE -HMA to AVR-PikC (and also to AVR-PikF). By contrast, Pikp SNK-EKE -HMA bound to both AVR-PikC and AVR-PikF with higher apparent affinity (larger %Rmax) than Pikp NK-KE -HMA (Figure 3a-b). This result was consistent across the three concentrations investigated, though binding (and %Rmax) was low for all three HMA domains at 4 nM ( Figure 3-figure supplement 1).
The Ser258Glu mutation extends the binding of Pikp-HMA NK-KE to AVR-PikC and AVR-PikF in planta Next, we determined whether the Ser258Glu mutation also extends binding to AVR-PikC and AVR-PikF in the full-length NLR in planta using co-immunoprecipitation. Full-length Pikp-1, Pikp-1 NK-KE , and                                               Pikp-1 SNK-EKE were each co-expressed with either AVR-PikD, AVR-PikC, or AVR-PikF in N. benthamiana. As before, Pikp-2 was not included in the co-immunoprecipitation assays to prevent the onset of cell death. We found that AVR-PikD, AVR-PikC, or AVR-PikF co-immunoprecipitated with Pikp-1 SNK-EKE ( Figure 3c); while the band corresponding to AVR-PikF was faint, this can be attributed to lower levels of AVR-PikF in the input. As previously observed (De la Concepcion et al., 2019), AVR-PikD, but not AVR-PikC or AVR-PikF co-immunoprecipitated with Pikp-1 and Pikp-1 NK-KE (Figure 3c). Taken together, the results from in vitro and in planta assays indicate that the Ser258Glu mutation increases the binding of Pikp-1 NK-KE for AVR-PikC and AVR-PikF to a sufficient level to trigger cell death in planta.

Crystal structures of the Pikp-HMA SNK-EKE /AVR-PikC and Pikp-HMA SNK-EKE /AVR-PikF complexes reveal new contacts across the binding interface
To confirm that the side chain of Glu258 in Pikp-1 SNK-EKE forms a new hydrogen bond across the interface (as observed for Glu72 in the OsHIPP19/AVR-PikF complex), we determined the crystal structures of the Pikp-HMA SNK-EKE /AVR-PikC and Pikp-HMA SNK-EKE /AVR-PikF complexes. For comparison, we also determined the crystal structure of Pikp-HMA NK-KE /AVR-PikC. These were produced by co-expression in E. coli and purified to homogeneity using established methods for purification of HMA domain/AVR-Pik complexes Maqbool et al., 2015;De la Concepcion et al., 2018;De la Concepcion et al., 2019). Crystals were obtained in several conditions in the Morpheus screen (Molecular Dimensions), and X-ray diffraction data were collected at the Diamond Light Source immunoprecipitation show that the Pikp-1 SNK-EKE chimera binds to tested AVR-Pik effector variants in N. benthamiana. Plant cell lysates were probed for the expression of Pikp-1/Pikp-1 NK-KE /Pikp-1 SNK-EKE and AVR-Pik effector variants using anti-FLAG and anti-Myc antiserum, respectively. Total protein extracts were visualized by Ponceau Staining. (D) The crystal structure of the Pikp-HMA SNK-EKE /AVR-PikC complex (PDB entry 7QPX) reveals additional hydrogen bonds at the protein:protein interfaces compared to Pikp-HMA NK-KE /AVR-PikC (PDB entry 7A8W). Protein structures are represented as ribbons with relevant side chains displayed as cylinders. Dashed lines indicate hydrogen bonds. Relevant water molecules are represented as red spheres.
The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Unedited and uncropped blot for Figure 3C, IP Pik-1 α-FLAG, Pik-1 α-FLAG, with relevant bands labeled.

Source data 10. Unedited and uncropped blot for
Source data 11. Surface plasmon resonance (SPR) data used for box plots in panel A.
The global structures of the complexes are essentially identical to each other and to the previously determined Pik-HMA/AVR-Pik crystal structures (Figure 3-figure supplement 2, Figure 3-figure  supplement 3, Figure 3-figure supplement 4, Supplementary file 1). The RMSDs, as calculated in COOT with secondary structure matching, between Pikp-HMA NK-KE /AVR-PikC and Pikp-HMA SNK-EKE / AVR-PikC or Pikp-HMA SNK-EKE /AVR-PikF are 0.38 Å using 154 residues and 0.60 Å using 155 residues, respectively. Interface analysis performed with qtPISA (Krissinel and Henrick, 2009) identified 15 hydrogen bonds and nine salt bridges between Pikp-HMA SNK-EKE and AVR-PikC, and 16 hydrogen bonds and 11 salt bridges between Pikp-HMA SNK-EKE and AVR-PikF, compared to the 12 hydrogen bonds and eight salt bridges mediating the interaction between Pikp-HMA NK-KE and AVR-PikC (Supplementary file 2). Inspection of the structures revealed that the side chain of Glu258 does indeed extend across the interface, forming direct hydrogen bonds with the backbone of the effectors (Figure 3d). This single mutation also supports additional hydrogen bonds at the interface between AVR-PikC (or AVR-PikF) and residues comprising β4 of the HMA domain (Figure 3d). These differences at interface 3 likely explain the increased binding affinity of Pikp-HMA SNK-EKE for AVR-PikC/AVR-PikF relative to Pikp-HMA NK-KE .

Discussion
Plant diseases cause significant crop losses and constrain global food production. To develop diseaseresistant crops, breeding programs exploit resistance genes present in wild germplasm that can be introgressed into elite cultivars. While recent advances have accelerated efforts to identify and clone resistance genes (Mascher et al., 2014;Arora et al., 2019;Jupe et al., 2013;Gardiner et al., 2020), conventional breeding approaches are constrained by the recognition profiles of resistance genes present in wild germplasm. Rational engineering of NLR immune receptors has the potential to yield novel disease resistance traits and expand the repertoire of resistance genes available to combat plant pathogens. It also offers the potential to restore disease resistance that has been overcome by pathogens and accelerate responses to dynamic changes in pathogen-effector populations. Here, we took two approaches to engineer the integrated HMA domain of the NLR protein Pik-1 to deliver new-to-nature effector recognition profiles.
The stealthy effector variants AVR-PikC and AVR-PikF do not interact with the integrated HMA domains of any Pik alleles characterized to date with sufficiently high affinity to activate defense. By contrast, as a putative virulence target of AVR-Pik, OsHIPP19 is bound by all effector variants, including AVR-PikC and AVR-PikF with high affinity Oikawa et al., 2020). Using this knowledge, and the relationship between OsHIPP19 and integrated Pik-HMA domains, we engineered two Pik-1 variants, Pikp-1 OsHIPP19-mbl7 , and Pikp-1 SNK-EKE . These engineered Pik-1 proteins bound AVR-PikC and AVR-PikF, activated cell death in N. benthamiana, and conferred blast resistance in rice. Engineering an NLR-integrated domain to resemble an effector target reduces the likelihood of the effector mutating to evade immune detection while retaining host target binding. Therefore, this approach may represent a route to more durable disease resistance, particularly in the case of effectors whose function is essential for pathogen virulence.
Despite the structural similarity of the OsHIPP19 and Pikp-1 HMA domains, the Pikp-1 OsHIPP19 chimera triggered effector-independent cell death in N. benthamiana when expressed with Pikp-2. This autoactivity required intact P-loop and MHD motifs in Pikp-2, as previously observed for the effector-dependent response of Pikp-1/Pikp-2 to AVR-PikD (Zdrzałek et al., 2020). Interestingly, mutating the conserved lysine in the P-loop of Pikp-1 OsHIPP19 to arginine partially attenuated the cell death response. Mutating the P-loop of Pikp-1 has previously been shown to abolish the effectordependent cell death response; however, maintenance of effector-independent cell death, albeit at a reduced level, by Pikp-1 OsHIPP19_K296R , suggests that an intact P-loop in Pikp-1 is not essential for Pik-mediated signaling. Based on a previously published approach to remove autoactivation when incorporating different HMA domains into Pik-1 (Białas et al., 2021), we reverted seven amino acids in the β1-α1 loop of Pikp-1 OsHIPP19 to those found in Pikp-1, giving the modified chimera Pikp-1 OsHIPP19-mbl7 . The β1-α1 loop contains the classical MxCxxC metal-binding motif which is characteristic of HMA domains and is degenerate in both Pikp-1 (MEGNNC) and OsHIPP19 (MPCEKS). We speculate that this loop is involved in intra-or inter-molecular interactions of Pik-1/Pik-2 which support an inactive state in the absence of effector binding, which is disturbed in the Pikp-1 OsHIPP19 chimera but restored in Pikp-1 OsHIPP19-mbl7 . These results highlight the potential challenges of incorporating domains that have not co-evolved with other domains in the receptor, but also show the potential for overcoming autoactivation to deliver functional NLRs. A recent study demonstrated that the Pik-1 chassis can accommodate nanobodies to GFP and mCherry, and these mediated reduced viral loads of Potato Virus X (PVX) expressing these antigens in N. benthamiana (Kourelis et al., 2023). This further demonstrates the potential of the Pik-1 NLR as a versatile system for engineering disease resistance through domain exchange.
Based on the OsHIPP19/AVR-PikF complex , we incorporated a Ser258Glu point mutation in the Pikp-1 NK-KE background, generating a Pikp-HMA triple mutant, Pikp-1 SNK-EKE . By determining the crystal structures of the Pikp-HMA SNK-EKE /AVR-PikC and Pikp-HMA SNK-EKE /AVR-PikF complexes, we confirmed the formation of new contacts across the HMA/effector interface that likely account for the expanded recognition profile to AVR-PikC and AVR-PikF. In addition to a new hydrogen bond between the side chain of Pikp-1 SNK-EKE_Glu258 and the backbone of AVR-PikC, we observed two additional intermolecular hydrogen bonds formed between other amino acids at the interface. This extended hydrogen bonding is facilitated by a shift in β4 of the HMA domain towards the effector. Together with previous studies in the Pik-1/Pik-2 and RGA5/RGA4 systems (De la Concepcion et al., 2019;Cesari et al., 2022;Liu et al., 2021), our new results show the utility of points presented in the box plots are shown in Figure 4-figure supplements 2-4. Lowercase letters represent statistically significant differences (p<0.05, ANOVA with post-hoc Tukey HSD test). RT-PCR confirmed expression of transgenes in (A) is shown in Figure 4-figure supplement 1. The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. Lesion size data for pathogen growth on rice plants.     structure-guided approaches to engineering NLR integrated domains to extend binding to different effectors. While recent advances in protein structure modeling will support future engineering efforts, challenges remain in the accurate prediction of side-chain positions, and the effect of individual mutations, which will necessitate experimental determination of protein complexes to optimize intermolecular interactions.
The Pikp-1 SNK-EKE variant differs from the wild-type Pikh-1 allele in just two amino acid positions. Generating Pikp-1 SNK-EKE from Pikh-1 requires a maximum of four nucleotide substitutions, which can be achieved using precise base editing and prime editing technologies (Molla et al., 2021;Hua et al., 2022). In many countries, edited crop varieties which do not contain DNA from another species are not subject to restrictions beyond those required for conventionally bred crop varieties. Therefore, this work raises the exciting prospect of editing wild-type alleles of NLRs that have greater potential for deployment in the field than those incorporating entirely new protein domains or substantial sequence changes.
Given the limited number of M. oryzae effectors for which host targets have been identified, it is notable that three (AVR-Pik, AVR-Pia and AVR1-CO39) interact with HMA domains of HIPPs and/or HPPs Oikawa et al., 2020). The AVR-Pik-like effector APikL2, which is highly conserved across M. oryzae lineages with different grass hosts, also binds to the HMA domain of a HIPP (sHMA94 from Setaria italica) . Although interaction with AVR-Pik appears to stabilize HIPPs (Oikawa et al., 2020), at present, the consequences and significance for pathogen virulence of these effector/HMA interactions are unclear. Intriguingly, the potato mop-top virus movement protein has been shown to interact with NbHIPP26 (Cowan et al., 2018), and other HIPPs/HPPs have been described as host susceptibility factors (Fukuoka et al., 2009;Radakovic et al., 2018;Zschiesche et al., 2015) and may represent targets of effectors from other pathogens. This raises the possibility that incorporating different HMA domains into the Pik-1 chassis (with the mutations described here to prevent autoactivation, if necessary), could offer a suite of NLR proteins capable of recognizing as-yet unknown effectors from diverse pathogens. Alongside biochemical approaches to identify effector-target interactions, advances in structural modeling could also enable the identification of novel HMA-binding effectors. AVR-Pik, AVR-Pia, and AVR1-CO39 all share the conserved MAX structural fold (de Guillen et al., 2015), and effectors with a similar structural core may bind HMAs. For example, an integrated HMA domain has been engineered to respond to the MAX effector AVR-Pib (Liu et al., 2021), although it is yet to be demonstrated that this effector binds host HMA targets. Identification of specific effector/HMA pairs could guide Pik-1 engineering for the recognition of new pathogens, and potentially enable the design of synthetic HMA domains capable of binding to and recognizing a broad range of pathogen effectors.
Advances in our understanding of the molecular and structural basis of NLR activation have progressed efforts for the rational engineering of NLR proteins with altered recognition profiles. Previous studies have successfully engineered integrated domains to extend recognition capacities, though so far this has either resulted in the regeneration of resistance already conferred by other NLRs (De la Concepcion et al., 2019;Cesari et al., 2022;Liu et al., 2021) or provided recognition of a protein not present in the native pathogen (Kourelis et al., 2023). In this study, we use an effector target to guide the engineering of an integrated domain to deliver two engineered Pik-1 variants with new-to-nature effector recognition profiles. The chimeric NLR Pikp-1 OsHIPP19-mbl7 highlights the potential to incorporate diverse HMA domains without rendering the chimera autoactive. The triple mutant Pikp-1 SNK-EKE illustrates the benefit of structural/biochemical characterization of effector-target interactions to inform rational engineering. Crucially, both engineered NLR proteins deliver novel resistance in transgenic rice, and have potential for deployment in the field against M. oryzae isolates carrying the stealthy AVR-PikC and AVR-PikF alleles. This study demonstrates the value of targetguided approaches in engineering NLR proteins with new-to-nature recognition profiles. We propose that this approach could expand the 'toolbox' of resistance genes to counter the devastating impacts of plant pathogens on crop yields and global food security.

Gene cloning for protein expression in N. benthamiana
For protein expression in planta, full-length Pikp-1 NLRs containing the OsHIPP19 HMA domain (and Pikp-1 OsHIPP43-mbl7 ), the Pikp-1 SNK-EKE mutation (made by introducing the S258E mutation in the HMA domain by PCR), and other HMA domain mutations were assembled using Golden Gate cloning into the plasmid pICH47742 with a C-terminal 6xHis/3xFLAG tag. Expression was driven by the A. tumefaciens Mas promoter and terminator. Full-length wild-type Pikp-1, Pikp-1 NK-KE , Pikp-2, and AVR-Pik variants used were generated as described previously (Maqbool et al., 2015;De la Concepcion et al., 2018;De la Concepcion et al., 2021;Longya et al., 2019;De la Concepcion et al., 2019). All DNA constructs were verified by sequencing.
Gene cloning, expression, and purification of proteins for in vitro binding studies For SPR, Pikp-HMA NK-KE and Pikp-HMA SNK-EKE (residues Gly186 -Asp264) variants were cloned into pOPIN-M (generating a 3 C protease cleavable N-terminal 6xHis:MBP-tag). AVR-PikD, AVR-PikC, and AVR-PikF (residues Glu22 -Phe93) were cloned into pOPIN-E (generating a C-terminal non-cleavable 6xHis-tag, but also including a 3 C protease cleavable N-terminal SUMO-tag, as detailed previously Maqbool et al., 2015). The Pikp-HMA NK-KE and Pikp-HMA SNK-EKE proteins were expressed and purified using the same pipeline as described below for obtaining protein complexes for crystallization, whereas the effectors were retained on the second pass through the 5 ml Ni 2+ -NTA column (which served to remove the SUMO tag following 3 C cleavage) requiring specific elution with elution buffer (50 mM Tris-HCl pH 8.0, 50 mM glycine, 0.5 M NaCl, 500 mM imidazole, 5% (v/v) glycerol), followed by gel filtration using a Superdex 75 26/600 column equilibrated in running buffer (20 mM HEPES pH 7.5 and 150 mM NaCl). Proteins were concentrated and stored at -80°C for further studies.
In vitro protein-protein interaction studies: SPR SPR was performed using a Biacore T200 (Cytiva) at 25 °C and at a flow rate of 30 µl/min. The running buffer was 20 mM HEPES pH 7.5, 860 mM NaCl, and 0.1%(v/v) Tween20. Flow cell (FC) 2 of an NTA chip (GE Healthcare) was activated with 30 µl 0.5 mM NiCl 2 . 30 µl of the 6xHis-tagged effector (the ligand) was immobilized on FC2 to give a response of ~250 RU. The HMA domain (the analyte) was then flowed over both FC1 and FC2 for 360 s, followed by a dissociation time of 180 s. Three separate concentrations of each HMA were tested, 4 nM, 40 nM, and 100 nM. The NTA chip was regenerated after each cycle with 30 µl 0.35 M EDTA pH 8.0. The background response from FC1 (non-specific binding of the HMA domain to the chip) was subtracted from the response from FC2. To obtain %R max , the binding response (R obs ) was measured immediately prior to the end of injection and expressed as a percentage of the theoretical maximum response (R max ) assuming a 2:1 HMA:effector binding model for Pikp-HMA, Pikp NK-KE -HMA, and Pikp SNK-EKE -HMA calculated as follows: Data analysis and visualization was carried out in R v4.1.2 (R Core Development Team, 2018) using the packages dplyr (v1.0.9 [Wickham, 2022]) and ggplot2 (v3.3.6 [Wickham, 2016]).

N. benthamiana cell death assays
A. tumefaciens GV3101 (C58 (rifR) Ti pMP90 (pTiC58DT-DNA) (gentR) Nopaline(pSouptetR)) cells carrying relevant Pikp-1 constructs, were resuspended in agroinfiltration media (10 mM MES pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone) and mixed with A. tumefaciens GV3101 carrying Pikp-2, AVR-Pik effectors, and P19 at OD 600 0.4, 0.4, 0.6, and 0.1, respectively. 4-week-old N. benthamiana leaves were infiltrated using a needleless syringe. N. benthamiana plants were grown in a controlled environment room at 22 °C constant temperature and 80% relative humidity, with a 16 hr photoperiod, and were returned to the same room following infiltration. Leaves were collected at 5 dpi (days post infiltration) and photographed under visible and UV light. Images shown are representative of three independent experiments, with a minimum of ten repeats (leaves) in each experiment. The cell death index used for scoring is as presented previously (De la Concepcion et al., 2018). Data analysis and visualization were carried out in R v4.1.2 (R Core Development Team, 2018) using the packages dplyr (v1.0.9 [Wickham, 2022]) and ggplot2 (v3.3.6 [Wickham, 2016]). Relevant comparisons between conditions (i.e. combinations of NLRs and effectors) in cell death assays were made using estimation methods (Ho et al., 2019) using the package besthr (v0.2.0 [MacLean, 2022]). Mean scores were calculated for each condition and each biological replicate. These means were ranked, and a mean rank was calculated for each condition. Bootstrapping was then used to estimate the confidence interval of the mean rank of each condition. 1000 samples were drawn (with replacement) from the values present in the dataset, and the mean rank was calculated for each of these samples to give a distribution of mean rank estimates. The 2.5 and 97.5 quantiles were determined. Conditions are considered to be different if their means lie outside the 2.5 and 97.5 quantiles.

Confirmation of protein production in cell death assays by western blot analysis
Western blot analysis was used to confirm the presence of proteins in N. benthamiana during cell death assays. Three leaf discs were taken at 2 dpi, flash-frozen in liquid nitrogen, and ground to a fine powder using a micropestle. Leaf powder was mixed with 300 μl of plant protein extraction buffer (GTEN (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % v/v glycerol), 10 mM DTT, 2% (w/v) PVPP, 0.1% Tween-20, 1 x plant protease inhibitor cocktail (Sigma)). The sample was clarified by centrifugation (20,000 × g for 2 min at 4 °C, twice) and 40 μL of the resulting supernatant was added to 10 μL SDS-PAGE loading dye (RunBlue 4 x LDS sample buffer (Expedeon)), followed by incubation at 95 °C for 5 min. Samples were subjected to SDS-PAGE/western blot analysis to detect epitope-tagged proteins. Pikp-1, Pikp-2, and AVR-Pik effectors were detected by probing membranes with anti-FLAG-HRP (Generon, 1:10,000 dilution), anti-HA-HRP (Thermo Fisher Scientific, 1:3000 dilution) and anti-Myc-HRP (Santa Cruz, 1:5000 dilution) antibodies, respectively, and LumiBlue ECL Extreme (Expedeon). Membranes were also stained with Ponceau S to observe protein loading.
In planta protein-protein interaction studies: co-immunoprecipitation For co-immunoprecipitation assays, three leaves were harvested at 3 dpi and flash-frozen in liquid nitrogen. Leaf tissue was ground to a fine powder in liquid nitrogen using a pre-chilled pestle and mortar and resuspended in ice-cold plant protein extraction buffer (2 ml/mg of powder). Plant cell debris was pelleted by centrifugation at 4200 × g for 30 min at 4 °C and the supernatant was filtered through a 0.45 μm membrane. 20 μL of filtered extracts were combined with 5 μL SDS-PAGE loading dye as input samples. For immunoprecipitation, 1 mL of filtered protein extract was mixed with 40 μL of anti-FLAG M2 magnetic beads (Merck, formerly Sigma-Aldrich) (equilibrated in GTEN +0.1% Tween-20 prior to use) in a rotary mixer for 1 hr at 4 °C. The beads were washed 5 X in ice-cold IP buffer by separating the beads using a magnetic rack, and the proteins were eluted by resuspending the beads in 30 μL SDS loading buffer and incubating at 70 °C for 10 min. Following SDS-PAGE and transfer, membranes were probed with antibodies as above.

Rice transformation and confirmation of transgene expression
To generate constructs for rice transformation, Golden Gate Level 1 constructs encoding a hygromycin resistance cassette (35 S promoter/nos terminator), untagged Pikp-1/Pikp-1 OsHIPP19-mbl7 /Pikp-1 SNK-EKE with the NLR flanked by the 35 S promoter and terminator, and untagged Pikp-2 also flanked by the 35 S promoter and terminator were assembled by Golden Gate assembly into the Level 2 vector pICSL4723. The resulting Level 2 constructs contained the hygromycin resistance cassette, 35 S::Pikp-1/Pikp-1 OsHIPP19-mbl7 /Pikp-1 SNK-EKE , and 35 S::Pikp-2. These constructs were introduced into Agrobacterium tumefaciens (strain EHA105) and used for Agrobacterium-mediated transformation of Oryza sativa cv. Nipponbare follows the method of Okuyama et al., 2011. PCR confirmation of the presence of Pikp-1/Pikp-2 transgenes in T 1 progenies used two primer sets, Pikp-1 (F: TG ATCA AAGA CCAC TTCC GCGT TC +R: T GCTG CCCG CAAT GTTT TCAC TGC) and Pikp-2 (F: AT TGTA TATG TCAG CCAG AAAA TG +R: T CCTC AGGG ACTT GCTC GTCT AC) that amplify respectively. To confirm transgene expression, total RNA was extracted from leaves using an SV Total RNA Isolation System (Promega, WI, USA) and used for RT-PCR. cDNA was synthesized from 500 ng total RNA using a Prime Script RT Reagent Kit (Takara Bio, Otsu, Japan). RT-PCR was performed using three primer sets, Pikp-1 (F: TG ATCA AAGA CCAC TTCC GCGT TC +R: T GCTG CCCG CAAT GTTT TCAC TGC), Pikp-2 (F: AT TGTA TATG TCAG CCAG AAAA TG +R: T CCTC AGGG ACTT GCTC GTCT AC) and OsActin (F: C TGAA GAGC ATCC TGTA TTG +R: GAAC CTTT CTGC TCCG ATGG ) that amplify Pikp-1, and OsActin,respectively. Disease resistance/virulence assays in rice Rice leaf blade punch inoculation was performed using the M. oryzae isolates. A conidial suspension (3 × 10 5 conidia mL −1 ) was punch inoculated onto a rice leaf one month after seed sowing. The inoculated plants were placed in a dark dew chamber at 27 °C for 24 hr and then transferred to a growth chamber with a 16 hr light/8 hr dark photoperiod. Disease lesions were scanned at 7 days post-inoculation (dpi), and lesion size was measured manually using Image J software (Schneider et al., 2012). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.